Unexpected identification of obesity-associated mutations in LEP and MC4R genes in patients with anorexia nervosa

Mutations leading to a reduced or loss of function in genes of the leptin-melanocortin system confer a risk for monogenic forms of obesity. Yet, gain of function variants in the melanocortin-4-receptor (MC4R) gene predispose to a lower BMI. In individuals with reduced body weight, we thus expected mutations leading to an enhanced function in the respective genes, like leptin (LEP) and MC4R. Therefore, we have Sanger sequenced the coding regions of LEP and MC4R in 462 female patients with anorexia nervosa (AN), and 445 healthy-lean controls. In total, we have observed four and eight variants in LEP and MC4R, respectively. Previous studies showed different functional in vitro effects for the detected frameshift and non-synonymous variants: (1) LEP: reduced/loss of function (p.Val94Met), (2) MC4R: gain of function (p.Val103Ile, p.Ile251Leu), reduced or loss of function (p.Thr112Met, p.Ser127Leu, p.Leu211fsX) and without functional in vitro data (p.Val50Leut). In LEP, the variant p.Val94Met was detected in one patient with AN. For MC4R variants, one patient with AN carried the frameshift variant p.Leu211fsX. One patient with AN was heterozygous for two variants at the MC4R (p.Val103Ile and p.Ser127Leu). All other functionally relevant variants were detected in similar frequencies in patients with AN and lean individuals.

Table 1.Minor allele frequencies of the detected variants in LEP and MC4R.Here, all detected variants in the coding regions of LEP and MC4R are represented for the respective study groups.The alleles are assigned based on the forward strand.Additionally, the minor allele frequency of the non-Finnish European females in the Genome Aggregation Database (gnomAD 90 ) for each SNP is given.Some genetic variants are not represented in the used version (v2.1.1) of gnomAD (NA).11, homozygous wildtype; 12, heterozygous; 22, homozygous minor allele.gnomAD, Genome Aggregation Database; MAF, minor allele frequency; NA, not available.a One female patient with AN carried two heterozygous variants (rs2229616, p.Val103Ile and rs13447331, p.Ser127Leu).She had a BMI of 15.67 kg/m 2 at admission, increased weight during her in-patient treatment and was discharged with a BMI of 17.75 kg/m 2 .After 2.5 years, her BMI was 22.66 kg/m 2 .Her leptin levels were 1.66 µg/l at admission, while these increased during in-patient treatment to 5.59 µg/l at discharge.At the 2.5 year follow-up, her leptin levels were 43.31 µg/l.b The patient with AN carrying this variant heterozygously had a BMI of 13.54 kg/m 2 at admission which increased to 18.56 kg/m 2 at discharge.At the follow-up time point of 2.5 years, her BMI was 18.29 kg/m 2 .Her leptin levels were at admission: 0.85 µg/l, at discharge 10.6 µg/l and at the follow-up time point 6.61 µg/l.One variant led to a non-conservative amino acid exchange (rs17151919, p.Val94Met), while all others were synonymous variants (rs201523305, p.Cys7=; rs13306517, p.Gln25= and a variant without an rsID, p.Ser91=).The non-synonymous variant (rs17151919) was detected in the heterozygous state in an acutely ill female patient with AN (age: 34.12 years old; BMI: 15.94 kg/m 2 ; BMI-SDS LMS : − 3.62).In silico tools, like MutationTaster2021 40 and PredictSNP2 41 , hinted at an overall non-pathogenic effect.Yet, the resultant amino acid substitution was predicted to have a destabilizing impact on LEP (see Table 2).Previously, two studies have implied a role of the mutant allele (Met94; in mature protein this equals position 73; see Supplementary Fig. 1) in body weight regulation 42,43 .Met94 was already reported to be associated with lower leptin levels and a higher childhood BMI in individuals of African ancestry 42 .At baseline, each mutant allele added approximately 2.6 kg to the carrier's weight, while the weight increased to 4.8 kg at year 20 43 .No associations were detected in Europeans, as this variant was extremely rare in this population 42 .Functional in vitro analyses reported a decreased leptin secretion in human embryonic kidney 293 cells caused by the substitution of valine by methionine 42 .
We observed the non-synonymous variants rs2229616 (p.Val103Ile) and rs52820871 (p.Ile251Leu) in similar frequencies in our 462 patients with AN, and 445 healthy-lean controls (see Table 1).Within our control group, which included both sexes, variant rs52820871 (p.Ile251Leu) was detected four times in each sex, while variant rs2229616 (p.Val103Ile) was present in four males and nine females.Our in silico analyses hinted at a putative deleterious effect caused by rs2229616 without affecting splice sites and the protein's torsion, while rs52828071 Table 2.In silico analyses for the detected variants in LEP and MC4R.To predict putative effects of the mutant alleles, various in silico tools were applied.Putative impacts on splicing products were examined with the tool ESEfinder 83 .The general pathogenicity was predicted with MutationTaster2021 40 and PredictSNP2 41 .The latter being a consensus classifier condensing results of multiple tools, like CADD and FATHMM.Indications for impacts on the proteins were investigated with PANTHER-PSEP 84 , CUPSAT 85 and SIFT 86 .Tools predicting effects on the protein were only applicable for non-synonymous variants.Yet, as the used protein reference sequence of MC4R was incomplete, the non-synonymous variant rs13447329 (p.Thr112Met) could not be analysed with CUPSAT and SIFT.was predicted to be overall benign but has the potential to destabilize MC4R (see Table 2).In our literature search, we found numerous studies reporting a reduced risk for obesity and an association with a lower BMI of the infrequent alleles of rs2229616 and rs52820871 21,22,44 .In 2004, Geller et al. 21detected a reduced transmission rate of the Ile103 allele in an analysis of family-based samples, thus providing initial evidence for an obesity-protective effect of this variant.Functional implications of both frequent variants have been thoroughly studied, as well.For instance, the introduction of the variants in inbred mice, showed no significant difference in food intake and BMI between homozygous or heterozygous mice for each mutant and their wildtype (WT) littermates.Yet, homozygous female mice (MC4R V103I/V103I and MC4R I251L/I251L ) had a reduced longitudinal length and their white adipose tissue weighed less in relation to their WT littermates.Albeit, when fed a high-fat diet, these mice's adiposity resembled those of their littermates not carrying a variant 23 .Another study showed that variants p.Val103Ile and p.Ile251Leu can be characterised as GoF variants as they have a significant bias towards the ß-arrestin recruitment rather than cyclic adenosine monophosphate (cAMP)-mediated signalling.Beyond, it was determined that the MC4R carrying the mutant Ile103 allele did not internalize like the WT MC4R and consequently exhibited a stable expression on the cell surface upon agonist stimulation 22 .In accordance, another study supported a deviating signalling behaviour due to p.Val103Ile yielding in GoF characteristics 45 .Conflicting to these findings, others reported that both variants displayed an elevated presence at the plasma membrane.Upon agonist stimulation, solely p.Ile251Leu led to a reduced internalisation, while the MC4R with the mutant Ile103 allele maintained an unaltered internalisation 46 .Besides, multiple studies did not ascertain any functional differences between the WT and mutant MC4R for p.Val103Ile and p.Ile251Leu 21,[47][48][49][50] .
In our mutation screen, we have further detected one lean control individual (BMI: 17.00 kg/m 2 ; BMI-SDS: − 2.64) harbouring two frequent variants leading to p.Val103Ile and p.Ile251Leu.Similarly, one patient with AN carried the common rs2229616 (p.Val103Ile) and the rare variant rs13447331 (p.Ser127Leu).This patient had a restrictive AN.At admission the patient had a weight of 45.3 kg being 169.5 cm tall (BMI: 15.67 kg/m 2 ; 4th BMI-percentile).During the 19 weeks in-patient treatment 6 kg were gained resulting in a BMI of 17.75 kg/m 2 at discharge.Leptin levels were 1.66 µg/l at admission and increased during treatment up to 5.59 µg/l at discharge.After 2.5 years, the patient had a BMI of 22.66 kg/m 2 (weight: 66 kg), while leptin levels were 43.31 µg/l.In vitro studies identified in the subsequent literature search on double mutant receptors with p.Val103Ile and p.Ser127Leu demonstrated that these can cause a reduced efficacy and potency 51 and are less abundant on cell surfaces 52 .An increase in the cAMP accumulation in relation to the single mutant MC4R carrying the Leu127 allele was determined 52 .Within our mutation analysis, we did not detect the rare variant leading to the nonconservative amino acid substitution from serine to leucine at position 127 in any additional individuals here (see Table 2).Our in silico analyses detected functional implications for this variant, as it was rated as deleterious by MutationTaster2021 and PredictSNP2.Yet, no destabilizing potential on the MC4R protein was indicated (see Table 2).
The frameshift mutation in MC4R (rs13447338) resulting in a truncated protein (p.Leu211fsX) was detected once in a female adolescent with AN, and not in the lean individuals (see Table 1).The female patient with AN displayed the restrictive form of AN.At admission, she weighed 35.10 kg while being 161 cm tall (BMI: 13.54 kg/ m 2 ; 0th BMI-percentile).During her 20-week in-patient treatment, she gained 13 kg.At discharge, she had a weight of 48.1 kg and a BMI of 18.56 kg/m 2 .Leptin levels were 0.85 µg/l at admission and increased to 10.6 µg/l at discharge.After 2.5 years, the patient's weight was 49 kg yielding in a BMI of 18.29 kg/m 2 , while leptin levels were 6.61 µg/l.One in vitro study validated the functional impact of the truncated protein reporting that this variant caused a complete loss of function 47 .
The other two non-synonymous and rare variants rs121913557 (p.Val50Leu) and rs13447329 (p.Thr112Met) were detected in both study groups.While in silico analyses hinted at a putative pathogenic and protein destabilizing impact of rs121913557, no functional implications for rs13447329 were detected (see Table 2).Previous studies analysing rs13447329 effects in vitro resulted in ambiguous findings.Thus, increased agonist potency and affinity for α-MSH were detected 53,54 .The mutant receptor further showed a decrease in cell surface expression in comparison to the WT 53 .Yet, multiple studies reported no functional difference of the mutant MC4R caused by this variant and the WT receptor 48,51,55 .We could not detect any studies in the evaluated literature resources, which have performed functional analyses for the Leu50 allele of rs121913557 so far (see Supplementary Tables 3, 4 and 5).
Both synonymous variants identified (p.Tyr153= and p.Val193=) were detected each in one proband (see Table 1).The variant p.Val193= was detected once in the lean control group and was implied to be benign (see Table 2).The other synonymous and novel variant p.Tyr153= was found in one patient with AN (BMI: 17.29 kg/ m 2 ).For this variant, in silico tools revealed a high capacity to affect splicing and to be pathogenic (see Table 2).Within a literature search, no previously published functional in vitro studies were available (see Supplementary Tables 3, 4 and 5).

Discussion
Genetic variants in genes of the leptin-melanocortin system, like LEP and MC4R, are predominantly associated with monogenic forms of obesity 13,15,18,20,36,39,[56][57][58] .Reports emerged indicating that some variants in these genes also predispose to a lower BMI 21,22,59 .Genetic correlations between BMI, obesity and AN have been uncovered 9,[24][25][26] .In fact, genetic variants for AN are negatively correlated with BMI, leptin levels, body fat percentage, waist circumference, overweight as well as obesity 9,26 .Previously, we have identified nine SNPs at three independent genetic loci being associated with low BMI and AN 24 .To expand the findings of our previous mutation screens 36,39 , we have sequenced the coding regions of LEP and MC4R in 462 female patients with AN, and 445 healthy-lean controls.Collectively, we have detected four variants in the LEP gene and eight variants in MC4R.Strikingly, we detected known variants leading to reduced or even loss of function which are associated www.nature.com/scientificreports/with obesity 39,60 .Yet, the MC4R-located variants rs2229616 (p.Val103Ile) and rs52820871 (p.Ile251Leu) detected in multiple patients with AN and healthy-lean controls were already known to be associated with a lower BMI and a reduced risk for obesity 21,22,44,61 .One GWAS for BMI performed for both sexes combined and separately revealed that rs2229616 (p.Val103Ile) was genome-wide significantly associated with the BMI in both sexes 38 .Thus confirming one of our previous studies 21 .In silico analyses indicated largely benign effects, while certain variants in both genes might impact the stability or torsion of the respective protein.
The relevance of the leptin-melanocortin system in body weight regulation, energy expenditure as well as AN is already known 10,29 .Patients with AN have reduced serum leptin levels, which typically rise as weight is restored [27][28][29] .Strikingly, low leptin levels were linked to AN hallmark characteristics, like hyperactivity and amenorrhea 62 .A recent Mendelian randomization study has reported that low leptin levels are correlated with a higher risk of AN.This correlation was also evident when analysing an exclusively female dataset.Notably, a correlation of AN with leptin levels was not detected 31 .Beyond, off-label treatment of patients with AN (case studies) with recombinant leptin helped to improve cognition, emotions, and behavioural traits, like hyperactivity and repetitive thoughts of food [32][33][34] .However, it remains uncertain how variants in the leptin gene have an effect on the etiology of AN.The here detected variant leading to the amino acid exchange of p.Val94Met was already linked to lower leptin levels 42 , which are known to increase AN risk 31 .Therefore, one could hypothesise that depending on other genetic variants or environmental exposures, one person may develop AN while another develops obesity.Yet, if this variant had an effect on body weight or reduced body weight, we would expect it to be equally present in underweight but healthy controls and not to be associated with obesity.Given that AN and obesity share some comorbidities, such as major depressive disorder [63][64][65] as well as body dissatisfaction 1,66,67 and regarding that metreleptin treatment in patients with AN and in one female with congenital leptin deficiency had beneficial effects on the mental traits [32][33][34]68 , the LEP variant might impact mental factors relevant for both, AN and obesity. Ye, further research is needed.
Evidence for the involvement of leptin's downstream target MC4R in AN is mostly based on animal models.In fact, the activation of MC4R in rats decreased food intake, while coincidently activating the hypothalamic-pituitary-adrenal axis.This activation eventually led to increased motor activity.It was suggested that genetic variants might trigger this coherence and thus lead to a prolonged stimulation of the leptin-melanocortin-system supporting the development of AN 5 .Yet, especially as overlaps between genetic loci relevant for low BMI and AN are known 24,25 , it is feasible that genetic variants in genes of the leptin-melanocortin system, impact the etiology of AN.
However, to date, no significant associations of genetic variants in these genes with AN were reported 36,37 .Due to our still limited sample size and generally low allele frequencies of the here detected variants, we were also unable to report associations with AN.Nevertheless, this study was able to expand our previously published mutation screens for both genes 36,39 .Our extended sample size enabled us to discover new variants in LEP and MC4R, which potentially are relevant for AN.This emphasises the ongoing necessity for comprehensive largescale genetic studies.Here, we did detect variants in LEP and MC4R, which were previously described to cause a partial or even complete loss of function in humans when present in a homozygous state and were thus associated with severe forms of obesity 39,51,60 .For instance, in 1999, we have described the frameshift mutation (rs13447338) yielding in a truncated MC4R in one adolescent and her mother with severe obesity 39 .In the present study, we have detected the same frameshift variant in the heterozygous state in one patient with AN.Yet, as no premorbid weight data was available, it is feasible that this patient had premorbid obesity triggering the development of AN.Generally, 2-5% of obesity cases are attributable to MC4R variants 13 .For the here commonly detected MC4R variants, like p.Val103Ile which was detected in 2% of patients with AN and 1.5% of controls, similar or slightly lower frequencies have been reported in patients with obesity 21,61,69,70 .Interestingly, this frequency is lower in females with obesity than in males with obesity 61 .The p.Val103Ile comprises the first polygenic variant for body weight regulation.The infrequent allele is associated with a lower BMI 21 .Further, we have to note that pathogenic variants typically are present homozygously 15,71 .For heterozygous MC4R variants, carrier develop obesity later in childhood 13 , while for heterozygous LEP variants, carriers are mainly unaffected 15,71 .As we have exclusively detected heterozygous variants in both genes, the relevance remains unknown.Yet, one patient with AN was detected to harbour two variants simultaneously (rs2229616, p.Val103Ile and rs13447331, p.Ser127Leu).Leptin levels at admission were in the expected range 72 and increased during in-patient treatment.Yet, after 2.5 years, leptin levels were rather high (43.31µg/l).The variant leading to p.Val103Ile is known to lead to an elevated function 21,22,46 , while the amino acid substitution of serine to leucine at the 127 th position was described to lead to a LoF in the majority of parameters analysed 45,55,73 (see Supplementary Tables 3, 4 and 5).Notably, certain studies also indicated no functional deviations for mutant receptors with either Ile103 or Leu127 48,49,54 .Yet, functional studies for a double mutant MC4R showed for example a reduced efficacy and potency 51 .Previously, we have also detected weight reducing variants in MC4R in patients with bulimia nervosa 74 .Further, the FTO variant rs9939609 associated with obesity 75,76 was previously identified in patients with eating disorders 77,78 .Besides a nominal association with bulimia nervosa and anorexia nervosa 77 , a synergic effect of this variant on leptin levels with an another variant in ABCA1 was described 78 .
In addition, a number of studies demonstrated that two types of MC4R variants exist which can be distinguished according to their distinct signalling behaviour 22,46 .For example, variants such as the rs13447329 (p.Thr112Met) detected here, are characterised by a LoF.Other variants, such as rs2229616 (p.Val103Ile), show a GoF phenotype 22,46 .The latter GoF variants are associated with a reduced risk for obesity and a lower BMI 21,22,44 .In fact, heterozygous carriers of GoF variants weighed on overage 0.39 kg/m 2 less than non-carriers, while homozygotes even showed an average 0.88 kg/m 2 decreased BMI 22 .Similarly, an animal model with transgenic mice expressing either Val103Ile or Ile251Leu exhibited GoF phenotypes.This was even more pronounced in female than male mice.For instance, female mice carrying the variant Val103Ile homozygous had a 40% lower abdominal white adipose tissue mass than their wildtype littermates.This difference was not detected in the male transgenic mice 23 .
Based on our data, we are unable to deduce a mechanism explaining how these variants may have a relevance for the phenotypes at the extremes of the weight scale.Yet, a resembling mechanism of action, as FTO's rs9939609 is feasible 78 .Functional in vitro and in vivo studies under fasting and starvation conditions are needed to expand the findings of the general in vitro data already published for each variant.
We are aware that in silico tools have their limitations and can vary substantially in their specificity [79][80][81] .Particularly, predicting functional implications of GoF variants was found to be more challenging by various in silico tools than finding an accurate implication for a LoF variant 82 .Hence, we have additionally checked the already published in vitro and in vivo data stated in LitVar 2 and on the www.mc4r.org.uk website.Nevertheless, no clear-cut results were obtained for most of the variants (see Supplementary Tables 2, 3, 4 and 5).Presumably, this is due to variations in study design as well as the time period in which the studies were conducted.For example, older studies increasingly failed to detect any differences between WT and mutant MC4R.This was particularly evident for studies investigating the common GoF variant rs2229616 (p.Val103Ile) in MC4R.Supposedly, this results from the technical advances and new experimental techniques implemented in recent years.In addition, there are many variants for which no or only very few functional studies have been completed.For example, only 16 studies have been conducted on the rs17151919 variant in the leptin gene, of which just one included functional in vitro analyses (see Supplementary Table 2).Conversely, for the common variant rs2229616 in MC4R, there are over 150 studies, of which numerous also present in vitro or even in vivo data (see Supplementary Tables 3, 4 and 5).
Given the reported genetic overlaps between BMI and AN 9,[24][25][26] and GoF variants in LEP and MC4R being associated with a lower BMI and a reduced odds for obesity [21][22][23]59 , we have Sanger sequenced the coding regions of LEP and MC4R in 462 female patients with AN and 445 healthy-lean controls. Wehave detected four variants in LEP and eight variants in the coding sequence of MC4R.Here we detected variants with a partial or complete LoF in vitro in heterozygous patients with AN.Homozygous carriers of these variants typically develop obesity.

Study groups
To detect variants in the coding regions of LEP and MC4R, 462 female patients with AN (acutely ill or recovered; age: 20.83 ± 8.63 years old; BMI: 16.48 ± 2.77 kg/m 2 ; BMI-SDS LMS : − 2.55 ± 1.58; see Table 3) and 445 healthy-lean individuals without a diagnosed eating disorder (age: 26.04 ± 5.77 years old; BMI: 18.08 ± 1.13 kg/m 2 ; BMI-SDS LMS : − 2.77 ± 0.58; see Table 3) were included.In previous studies we have already screened 49 patients with AN for LEP and 51 patients with AN and 25 lean individuals for MC4R 36,39 .Written informed consent was given by all participants and in case of minors by their parents.This study was approved by the Ethics committee of the respective Universities and was performed in accordance with the Declaration of Helsinki.

Hardy-Weinberg-Equilibrium
All detected variants were checked for compliance with the Hardy-Weinberg-Equilibrium (HWE).All variants fulfilled the HWE.

In silico analyses and assessment of functional implications by published data
To uncover putative functional implications of the detected variants, in silico analyses applying various tools ensued.The detected variants were analysed pertaining their potential to alter splice sites (ESEfinder 83 ) and their general pathogenicity (MutationTaster2021 40 ; PredictSNP2 41 ).Effects of non-synonymous variants on the protein structure of LEP or MC4R were examined using PANTHER-PSEP 84 , the Cologne University Protein To extend the functional implications derived from the in silico analyses with previously published in vitro and in vivo data, the LitVar 289 (accessed on Sep.26th 2022) database was screened for each variant in LEP and MC4R detected in this study.For MC4R variants, the website www.mc4r.org.uk (accessed on Sep.26th 2022) summarizing publications for each variant, was additionally analysed.This website is provided by the University of Cambridge Metabolic Research Laboratories.A complete list of all studies provided by LitVar 2 and www.mc4r.org.uk can be found in the Supplementary Table 5. https://doi.org/10.1038/s41598-024-57517-w

Table 3 .
Phenotypic characteristics of the investigated study groups.All data is represented as mean (SD).BMI, body mass index; BMI-SDS LMS , body mass index standard deviation score; SD, standard deviation.

in years BMI in kg/m 2 BMI-SDS LMS
88ability Analysis Tool85(CUPSAT) and the tool Sorting from Intolerant to Tolerant 86 (SIFT).The required protein structures were extracted from the Research Collaboratory for Structural Bioinformatics protein data bank (RCSB PDB): LEP (1AX887) and MC4R (7AUE88).